JP6341486B2 - Composition from lobster blood cell extract for detecting lipopolysaccharide, peptidoglycan and 1,3-β-D-glucan - Google Patents
Composition from lobster blood cell extract for detecting lipopolysaccharide, peptidoglycan and 1,3-β-D-glucan Download PDFInfo
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- JP6341486B2 JP6341486B2 JP2014549358A JP2014549358A JP6341486B2 JP 6341486 B2 JP6341486 B2 JP 6341486B2 JP 2014549358 A JP2014549358 A JP 2014549358A JP 2014549358 A JP2014549358 A JP 2014549358A JP 6341486 B2 JP6341486 B2 JP 6341486B2
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- peptidoglycan
- glucan
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- lipopolysaccharide
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Description
本発明は、特に微生物及びそれらの細胞壁に関連する構造、例えばグラム陰性菌からのリポ多糖（ＬＰＳ）、すなわち内毒素、グラム陰性菌及び陽性菌からのペプチドグリカン（ＰＧ）、並びに菌類及び酵母からの１，３−β−Ｄ−グルカン（ＢＧ）などの検出を伴う化学、薬剤学及び生物工学の分野に関する。本発明はまた、半導体産業において、及び臨床検査のための診断ツールとして使用されるような超純水中の微生物汚染の評価における用途を有する。 The invention particularly relates to structures from microorganisms and their cell walls, such as lipopolysaccharide (LPS) from gram-negative bacteria, ie endotoxins, peptidoglycans (PG) from gram-negative and positive bacteria, and fungi and yeast. It relates to the fields of chemistry, pharmacology and biotechnology with the detection of 1,3-β-D-glucan (BG) and the like. The present invention also has applications in the assessment of microbial contamination in ultrapure water such as used in the semiconductor industry and as a diagnostic tool for clinical testing.
自然免疫系は、多数の細菌によって保存かつ共有される分子構造の認識を介した微生物を検出するための非常に効果的な応答機構を提供する。病原体関連分子パターン（ＰＡＭＰ）として知られているこれらの構造は、宿主においては見出されず、かつ微生物の生存又は病原性において必須である。最も特徴的なＰＡＭＰは中でも、とりわけ、ＬＰＳ、ＢＧ及びＰＧである［Ｍｅｄｚｈｉｔｏｖ ａｎｄ Ｊａｎｅｗａｙ，Ｔｈｅ Ｎｅｗ Ｅｎｇｌａｎｄ Ｊｏｕｒｎａｌ ｏｆ Ｍｅｄｉｃｉｎｅ，３４３（５）：３３８−３４４，２０００（非特許文献１）］。 The innate immune system provides a highly effective response mechanism for detecting microorganisms through the recognition of molecular structures conserved and shared by many bacteria. These structures, known as pathogen-associated molecular patterns (PAMPs), are not found in the host and are essential for microbial survival or pathogenicity. Among the most characteristic PAMPs are, among others, LPS, BG and PG [Medshitov and January, The New England Journal of Medicine, 343 (5): 338-344, 2000].
内毒素は、その驚くべき生物学的能力、遍在性、従来の滅菌方法に対する耐性、及び非経口溶液を汚染する可能性の高さのために、薬剤学及び生物工学の産業において最も関連性のあるＰＡＭＰ又は外因性発熱物質であるということが長年にわたって確立されてきた［Ｗｉｌｌｉａｍｓ Ｋ．Ｌ．Ｅｎｄｏｔｏｘｉｎ Ｒｅｌｅｖａｎｃｅ ａｎｄ Ｃｏｎｔｒｏｌ Ｏｖｅｒｖｉｅｗ；ｉｎ Ｗｉｌｌｉａｍｓ，Ｋ．Ｌ．（ｅｄｓ）．Ｅｎｄｏｔｏｘｉｎｓ，Ｐｙｒｏｇｅｎｓ，ＬＡＬ Ｔｅｓｔｉｎｇ ａｎｄ Ｄｅｐｙｒｏｇｅｎａｔｉｏｎ．Ｔｈｉｒｄ ｅｄｉｔｉｏｎ．Ｈｅａｌｔｈｃａｒｅ Ｒｅｐｏｒｔ，Ｎｅｗ Ｙｏｒｋ，Ｌｏｎｄｏｎ．２００７，２７−４６（非特許文献２）］。さらに、内毒素は、グラム陰性菌によって引き起こされる敗血症と関連する内毒素ショックの主な原因であり、かつ自然免疫系に対してこれらの細菌の存在を示す最初の警報シグナルを構成する。ＬＰＳの血流中へのアクセスにより、全身性炎症反応症候群（ＳＩＲＳ）、多臓器不全（ＭＯＦ）、ショック及び死亡など、人間の健康に重大な悪影響が生じる［Ｏｐａｌ ＳＭ，Ｃｏｎｔｒｉｂ Ｎｅｐｈｒｏｌ １６７，１４−２４，２０１０（非特許文献３），Ｈｏｄｇｓｏｎ ＪＣ，Ｊ Ｃｏｍｐ Ｐａｔｈｏｌ １３５，１５７−１７５，２００６（非特許文献４）］。 Endotoxin is most relevant in the pharmaceutical and biotechnology industries due to its amazing biological ability, ubiquity, resistance to conventional sterilization methods, and high potential for contaminating parenteral solutions. It has been established for many years to be some PAMP or exogenous pyrogen [Williams K. et al. L. Endotoxin Relevance and Control Overview; in Williams, K .; L. (Eds). Endotoxins, Pyrogens, LAL Testing and Depyrogenation. Third edition. Healthcare Report, New York, London. 2007, 27-46 (non-patent document 2)]. In addition, endotoxin is a major cause of endotoxin shock associated with sepsis caused by Gram-negative bacteria and constitutes the first alarm signal indicating the presence of these bacteria to the innate immune system. Access to the bloodstream of LPS causes serious adverse effects on human health such as systemic inflammatory response syndrome (SIRS), multiple organ failure (MOF), shock and death [Opal SM, Contrib Nephrol 167, 14- 24, 2010 (Non-Patent Document 3), Hodgson JC, J Comp Pathol 135, 157-175, 2006 (Non-Patent Document 4)].
カブトガニ血球抽出成分（ＬＡＬ）試験は、特に非経口薬剤及び生物工学製品の品質管理において、細菌内毒素を検出するために広く使用されている（米国特許第４１０７０７７号（特許文献１）、同第４２７９７７４号（特許文献２）、同第４３２２２１７号（特許文献３）、同第３９５４６６３号（特許文献４）、同第４０３８０２９号（特許文献５）、同第４１８８２６４号（特許文献６）、同第４５１０２４１号（特許文献７）、同第５３１０６５７号（特許文献８））。 The horseshoe crab blood cell extract (LAL) test is widely used to detect bacterial endotoxins, especially in the quality control of parenteral drugs and biotechnological products (US Pat. No. 4,107,077). No. 4279774 (Patent Literature 2), No. 4322217 (Patent Literature 3), No. 3954663 (Patent Literature 4), No. 4038029 (Patent Literature 5), No. 4188264 (Patent Literature 6), No. 4,510,241 (Patent Document 7) and No. 5,310,657 (Patent Document 8).
ＬＡＬ試薬はカブトガニの血リンパ中に存在するアメーバ様細胞の抽出物から調製される。これはトリプシン様セリンペプチダーゼのカスケードから成り、Ｃ因子及びＧ因子それぞれを介して内毒素及び（１，３）−β−Ｄ−グルカンの存在下において活性化される。試薬はカブトガニの自然免疫系の一部であるカブトガニの凝固反応に基づくものである。同様のシステムは、これまでのところ、他の無脊椎動物種において発見されていない［Ｉｗａｎａｇａ Ｓ ｙ Ｌｅｅ ＢＬ，Ｊ Ｂｉｏｃｈｅｍ Ｍｏｌ Ｂｉｏｌ ３８，１２８−１５０，２００５（非特許文献５）］。 The LAL reagent is prepared from an extract of amoeba-like cells present in the hemolymph of horseshoe crab. It consists of a cascade of trypsin-like serine peptidases and is activated in the presence of endotoxin and (1,3) -β-D-glucan via factor C and factor G, respectively. The reagent is based on the coagulation reaction of horseshoe crabs that are part of the innate immune system of horseshoe crabs. A similar system has not been found so far in other invertebrate species [Iwanaga Sy Lee BL, J Biochem Mol Biol 38, 128-150, 2005].
内毒素特異的ＬＡＬ試薬は、（１，３）−β−Ｄ−グルカンに対して感受性を有するＧ因子を分離して、ＬＡＬの酵素カスケードの残りの成分を残すことにより（米国特許第５４０１６４７号（特許文献９）、同第５６０５８０６号（特許文献１０）、米国特許出願公開第２００３０１０４５０１号（特許文献１１））、又はＧ因子の活性化を阻害することにより（米国特許第５０４７３５３号（特許文献１２）、同第５１５５０３２号（特許文献１３）、同第５４７９８４号（特許文献１４）、同第５７０２８８２号（特許文献１５）、同第５９９８３８９号（特許文献１６）、同第５１７９００６号（特許文献１７））得られる。 Endotoxin-specific LAL reagents isolate the Factor G that is sensitive to (1,3) -β-D-glucan, leaving the remaining components of the LAL enzyme cascade (US Pat. No. 5,401,647). (Patent Document 9), US Pat. No. 5,605,806 (Patent Document 10), US Patent Application Publication No. 20030104501 (Patent Document 11)), or by inhibiting the activation of factor G (US Pat. No. 5,047,353) 12), US Pat. No. 5,155,032 (Patent Document 13), US Pat. No. 5,479,984 (Patent Document 14), US Pat. No. 5,702,882 (Patent Document 15), US Pat. No. 5,998,389 (Patent Document 16), and US Pat. No. 5,179,006 (Patent Document). 17)) is obtained.
カブトガニは過去３億年でほとんど進化していないために生きた化石として知られている海洋節足動物である。類似の試薬が得られる４種のカブトガニが存在する。 Horseshoe crabs are marine arthropods known as living fossils because they have hardly evolved over the last 300 million years. There are four species of horseshoe crabs that provide similar reagents.
タキプレウス・トリデンタツス（Ｔａｃｈｙｐｌｅｕｓ ｔｒｉｄｅｎｔａｔｕｓ）、タキプレウス・ギガス（Ｔａｃｈｙｐｌｅｕｓ ｇｉｇａｓ）及びカルシノスコルピウス・ロツンディカウダ（Ｃａｒｃｉｎｏｓｃｏｒｐｉｕｓ ｒｏｔｕｎｄｉｃａｕｄａ）の種は、アジアのみに存在する。最後の２種は個体数が少なく、試薬の生産を持続したことがない。さらに、一般的に中国カブトガニ又は三棘（ｔｒｉ−ｓｐｉｎｅ）カブトガニとして知られているタキプレウス・トリデンタツスは、中国海岸に沿って、特に南シナ海北部及び海南島の範囲領域において、高い個体群密度を有していた。しかしながら、台湾、日本、香港、タイ、中国におけるＴ．トリデンタツスに関する最近の研究では、個体数はほぼ絶滅まで大幅に減少していることが示唆されている。主な原因は乱獲及び海洋汚染である。 The species of Tachypleus tridentatus, Tachypleus gigas and Carcinoscorpius rotundicauda are present only in Asia. The last two species have a small number of individuals and have never sustained reagent production. Furthermore, Tachypleus tridentatus, commonly known as Chinese horseshoe crab or tri-spine horseshoe crab, has a high population density along the coast of China, particularly in the northern South China Sea and Hainan Island. It was. However, in Taiwan, Japan, Hong Kong, Thailand, China Recent research on Tridentatus suggests that the population has declined substantially to extinction. The main causes are overfishing and marine pollution.
アジア種の個体群密度が低いために、販売されているＬＡＬ試薬は、主にアメリカカブトガニとしても知られているリムルス・ポリフェムス（Ｌｉｍｕｌｕｓ ｐｏｌｙｐｈｅｍｕｓ）から得られる。リムルスは北部のメイン州からユカタン半島及びメキシコ湾にわたって米国大西洋岸に生息している。最大の個体群はデラウェア湾で発見されたが、その数及び産卵活動は大幅に減少している［Ｗｉｄｅｎｅｒ ＪＷ ａｎｄ Ｂａｒｌｏｗ ＲＢ，Ｂｉｏｌｏｇｉｃａｌ Ｂｕｌｌｅｔｉｎ（１９７）：３００−３０２，１９９９（非特許文献６）］。主な原因は、生息地の減少及びカキやウナギ釣りでの餌としての使用である［Ｒｕｄｌｏｅ Ａ，Ｊｏｕｒｎａｌ ｏｆ Ｉｎｖｅｒｔｅｂｒａｔｅ Ｐａｔｈｏｌｏｇｙ（４２）：１６７−１７６，１９８３（非特許文献７）；Ｂｏｔｔｏｎ ＭＬ，Ｂｉｏｌｏｇｉｓｔ（４９）：１９３−１９８，２００２（非特許文献８）］。ＬＡＬ試薬を調製するための出血工程による死亡率は約１５％の範囲であったが［Ｗａｌｌｓ ＥＡ ａｎｄ Ｂｅｒｋｓｏｎ Ｊ，Ｖｉｒｇｉｎｉａ Ｊｏｕｒｎａｌ ｏｆ Ｓｃｉｅｎｃｅ ５１（３）：１９５−１９８，２０００（非特許文献９）］、過去から倍増しており、その個体群の減少を加速させている。 Due to the low population density of Asian species, the LAL reagent sold is obtained primarily from Limulus polyphemus, also known as the horseshoe crab. Limulus lives on the Atlantic coast of the United States from the northern Maine to the Yucatan Peninsula and the Gulf of Mexico. The largest population was found in Delaware Bay, but its number and spawning activity have been greatly reduced [Widerer JW and Barlow RB, Biological Bulletin (197): 300-302, 1999]. . Major causes are habitat loss and use as bait for oyster and eel fishing [Rudloe A, Journal of Invertebrate Pathology (42): 167-176, 1983 (Bonton ML, Biologist) (49): 193-198, 2002 (Non-Patent Document 8)]. Although the mortality due to the bleeding process for preparing the LAL reagent was in the range of about 15% [Walls EA and Berkson J, Virginia Journal of Science 51 (3): 195-198, 2000 (Non-patent Document 9)] Has doubled since the past and has accelerated the decline of its population.
ＦＡＯによると、ＬＡＬ試薬が高価格であり、カブトガニの分布範囲が狭く、及びそれらが性的成熟に到達するのに非常に長い時間がかかるために、カブトガニの個体数は回復率を下回って減少しやすい。４種は絶滅の危機に瀕しており、かつ国際自然保護連合（ＩＵＣＮ）のレッドリストに含まれている。 According to the FAO, the population of horseshoe crabs decreases below the recovery rate due to the high cost of LAL reagents, the narrow distribution range of horseshoe crabs, and the time it takes to reach sexual maturity. It's easy to do. Four species are on the verge of extinction and are on the Red List of the International Union for Conservation of Nature (IUCN).
カブトガニの個体群の現在の危機的な状況を考慮して、米国大西洋沿岸州海洋漁業委員会（ＡＳＭＦＣ）はリムルスの釣り及び利用に関するいくつかの側面を規制している。ＬＡＬ業界に関しては、検体を死亡に至らしめるように出血させることはできず、かつ７２時間未満以内にマークされた捕獲場所に健康な状態で戻さなければならないことを確立した。また、ＬＡＬを生産するために使用する動物の年間割当量が設定され、これらの取り扱いに関連する死亡率はこの割当量の１５％を超えることができない。 In view of the current critical situation of horseshoe crab populations, the US Atlantic Ocean Fisheries Commission (ASMFC) regulates several aspects of Limulus fishing and use. With regard to the LAL industry, it has been established that specimens cannot be bled to death and must be returned to a well-known capture location within less than 72 hours. Also, an annual quota for animals used to produce LAL is set, and the mortality associated with their handling cannot exceed 15% of this quota.
世界的に、原料供給源のリムルスからの血リンパが利用できなくなる前に、内毒素及び他の発熱物質を検出するための代替物を研究する必要性が高まってきている。個体数の減退と相まってその需要の増加が継続する一方で、ＬＡＬを生産するために業界で使用できるカブトガニの年間割当量の確立により、ＬＡＬ試薬を生産するためにカブトガニの捕獲及び出血の増加を維持することは不可能であることが示唆される。 Worldwide, there is a growing need to study alternatives for detecting endotoxins and other pyrogens before hemolymph from the source Limulus is unavailable. While the increase in demand continues with the decline in population, the establishment of annual quotas of horseshoe crabs that can be used in the industry to produce LAL will increase the capture and bleeding of horseshoe crabs to produce LAL reagents. It is suggested that it is impossible to maintain.
米国特許第４２２９５４１号（特許文献１８）、同第５０８２７８２号（特許文献１９）、同第６７９０６５９号（特許文献２０）及び米国特許出願公開第２００３０１８６４３２号（特許文献２１）では、ＬＡＬを調製するためのアメーバ様細胞の天然供給源であるカブトガニ血リンパの代替案として、アメーバ様細胞を生体外培養するための戦略開発を記載している。現在までに、この方法で得られる商業用試薬、又はそれを得るためのプロジェクトは存在していない。 US Pat. Nos. 4,229,541 (Patent Document 18), 50508278 (Patent Document 19), 6790659 (Patent Document 20) and US Patent Application Publication No. 20030186432 (Patent Document 21) are used to prepare LAL. Describes the development of a strategy for in vitro culture of amoeba-like cells as an alternative to horseshoe crab hemolymph, a natural source of amoeboid cells. To date, there are no commercial reagents obtained by this method, or projects to obtain it.
一方で、新たな方法論によって、シンガポールカブトガニのカルシノスコルピウス・ロツンディカウダからの内毒素感受性Ｃ因子における組換え体のクローン化及び生産に成功し（米国特許第５７１２１４４号（特許文献２２）、同第５８５８７０６号（特許文献２３）、同第５９８５５９０号（特許文献２４））、天然のＬＡＬ試薬と類似の手法でＬＰＳ、すなわち内毒素を検出及び定量化するためのその使用が確立されてきた（米国特許第６６４５７２４号（特許文献２５））。組換えＣ因子と界面活性剤との組み合わせ（米国特許出願公開第２００３００５４４３２号（特許文献２６））、又は増加した感度及び安定性を示す製剤中の組換えＣ因子若しくは天然供給源から精製されて得られた組換えＣ因子（米国特許出願公開第２００４０２３５０８０号（特許文献２７））の試薬製剤が特許を取得している。しかしながら、試薬中に天然に存在する他の酵素の欠如のために、これらの製剤は酵素カスケードの増幅効果により提供される感度を欠いていたが、それはより感受性のある蛍光基質を使用することによって部分的に解決された。凝固酵素（米国特許出願公開第２００９０２０８９９５号（特許文献２８））及びＧ因子（米国特許出願公開第２０１００１１２６６号（特許文献２９））などのＬＡＬカスケードの他の成分もまた組換え生産されている。 On the other hand, a new methodology has succeeded in cloning and producing a recombinant in endotoxin-sensitive factor C from Carcinoscorpius rotundicauda of Singapore horseshoe crab (US Pat. No. 5,712,144 (patent document 22), US Pat. No. 5,858,706). (Patent document 23) and US Pat. No. 5,985,590 (patent document 24), LPS, that is, its use for detecting and quantifying endotoxin in a manner similar to that of natural LAL reagent has been established (US Patent). No. 6645724 (Patent Document 25)). A combination of recombinant factor C and a surfactant (US Patent Publication No. 20030054432) or purified from recombinant factor C or natural sources in a formulation that exhibits increased sensitivity and stability The obtained reagent preparation of recombinant factor C (US Patent Application Publication No. 200402235080 (Patent Document 27)) has obtained a patent. However, due to the lack of other naturally occurring enzymes in the reagents, these formulations lacked the sensitivity provided by the amplification effect of the enzyme cascade, which was achieved by using a more sensitive fluorescent substrate. Partially resolved. Other components of the LAL cascade, such as clotting enzymes (US Patent Application Publication No. 20090209995) and Factor G (US Patent Application Publication No. 2010012662) have also been produced recombinantly.
ＬＡＬ試薬及び組換えＣ因子はＬＰＳを検出できるが、ＰＧを検出しない。しかしながら、最近の研究では、非経口使用のための製剤中、及び非発熱性でなければならない他の溶液及びデバイスにおいて、ＰＧ含有量の検出かつ制御においても緊急の必要性が示されている。ＰＧはグラム陽性菌及びグラム陰性菌の細胞壁のよく保存された成分であり、かつ敗血症性ショックを含む、グラム陽性菌によって引き起こされる炎症反応及びその有害な健康への影響に対する主な原因である［Ｓｉｌｈａｖｙ ＴＪ ｅｔ ａｌ．，Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ ｐｅｒｓｐｅｃｔｉｖｅｓ ｉｎ ｂｉｏｌｏｇｙ ２，ａ０００４１４，２０１０（非特許文献１０）］。ＬＰＳのように、ＰＧは、炎症誘発性サイトカインの放出を誘導することができ［Ｖｅｒｈｏｅｆ Ｊ ａｎｄ Ｍａｔｔｓｓｏｎ Ｅ，Ｔｒｅｎｄｓ ｉｎ Ｍｉｃｒｏｂｉｏｌｏｇｙ ３：１３６−１４０，１９９５（非特許文献１１）；Ｔｅｔｉ Ｇ，Ｔｒｅｎｄｓ ｉｎ Ｍｉｃｒｏｂｉｏｌｏｇｙ ７：１００−１０１，１９９９（非特許文献１２）］、発熱性であり、細菌の増殖及び死の間に環境中に放出され、かつ通常の滅菌手段に対して耐性を示す［Ｍｏｅｓｂｙ Ｌ，Ｅｕｒｏｐｅａｎ Ｊｏｕｒｎａｌ ｏｆ Ｐｈａｒｍａｃｅｕｔｉｃａｌ Ｓｃｉｅｎｃｅ ３５，４４２−４４６，２００８（非特許文献１３）］。ＬＰＳとＰＧとの類似性のために、双方に同じ重要性を付与することが提案されている［Ｍｙｈｒｅ ＡＥ ｅｔ ａｌ．，Ｓｈｏｃｋ ２５（３）：２２７−３５，２００６（非特許文献１４）］。
LAL reagent and recombinant factor C can detect LPS but not PG. However, recent studies have also indicated an urgent need for detection and control of PG content in formulations for parenteral use and in other solutions and devices that must be nonpyrogenic. PG is a well-preserved component of the cell walls of Gram-positive and Gram-negative bacteria and is a major cause for inflammatory reactions and their adverse health effects caused by Gram-positive bacteria, including septic shock [ Silhavey TJ et al. , Cold Spring Harbor perspectives in
さらに、ＰＧの存在から、相乗作用により［Ｔａｋａｄａ Ｈ ｅｔ ａｌ．，Ｊｏｕｒｎａｌ ｏｆ Ｅｎｄｏｔｏｘｉｎ Ｒｅｓｅａｒｃｈ ８，３３７−３４２，２００２（非特許文献１５）；Ｈａｄｌｅｙ ＪＳ ｅｔ ａｌ．，Ｉｎｆｅｃｔｉｏｎ ａｎｄ Ｉｍｍｕｎｉｔｙ，７３：７６１３−７６１９，２００５（非特許文献１６）；Ｗｒａｙ ＧＭ，Ｓｈｏｃｋ，１５（２）：１３５−４２，２００１（非特許文献１７）；Ｓｈｉｋａｍａ Ｙ ｅｔ ａｌ．，Ｉｎｎａｔｅ Ｉｍｍｕｎｉｔｙ，１７（１）：３−１５，２００９（非特許文献１８）］、又は相加効果により［Ｓｐｒｏｎｇ Ｔ，Ｊｏｕｒｎａｌ ｏｆ Ｌｅｕｋｏｃｙｔｅ Ｂｉｏｌｏｇｙ，７０：２８３−２８８，２００１（非特許文献１９）］、ＬＰＳによって誘導される炎症反応を顕著に感作することが可能である。 Furthermore, because of the presence of PG, synergistic effects [Takada H et al. , Journal of Endotoxin Research 8, 337-342, 2002 (Non-Patent Document 15); Hadley JS et al. , Infection and Immunity, 73: 7613-7619, 2005 (Non-Patent Document 16); Wray GM, Shock, 15 (2): 135-42, 2001 (Non-Patent Document 17); Shikama Y et al. , Innate Immunity, 17 (1): 3-15, 2009 (Non-patent Document 18)], or by an additive effect [Spron T, Journal of Leukocyte Biology, 70: 283-288, 2001 (Non-patent Document 19)] It is possible to significantly sensitize the inflammatory response induced by LPS.
カブトガニのＬＡＬカスケードと同様に、プロフェノールオキシダーゼ活性化系（ＰｒｏＰＯ系）は、無脊椎動物の体液性自然免疫応答の必須の構成要素である［Ｃｅｒｅｎｉｕｓ ｅｔ ａｌ．，Ｔｒｅｎｄｓ Ｉｍｍｕｎｏｌ．（２９）：２６３−２７１，２００８（非特許文献２０）］。バイオセンサを介してＰＡＭＰを認識し、かつプロフェノールオキシダーゼ活性化酵素（ｐｐＡ）又はフェノールオキシダーゼの酵素活性を測定することにより可視的かつ定量可能な応答をもたらすｐｒｏＰＯ系の能力により、それを微生物及びそのＰＡＭＰの検出用試薬を開発するための魅力的な候補とする。 Similar to the horseshoe crab LAL cascade, the prophenoloxidase activation system (ProPO system) is an essential component of the invertebrate humoral innate immune response [Cerenius et al. , Trends Immunol. (29): 263-271, 2008 (Non-Patent Document 20)]. The ability of the proPO system to recognize PAMP via a biosensor and produce a visible and quantifiable response by measuring the enzymatic activity of prophenoloxidase activating enzyme (ppA) or phenol oxidase makes it a microorganism and It is an attractive candidate for developing a reagent for detecting PAMP.
ＰｒｏＰＯ系は、パターン認識タンパク質、ｐｐＡ及びｐｒｏＰＯを含むタンパク質の複雑な配列を含む。いくつかの節足動物において、ｐｒｏＰＯ系はまだ完全に解明されていない機構を介して少量のＰＡＭＰの存在下で活性化されることが記載されている。一般には、ＰＡＭＰの存在下でｐｒｏ−ｐｐＡは活性となり、タンパク質分解攻撃によってプロフェノールオキシダーゼ（不活性酵素前駆体）を活性フェノールオキシダーゼに変換することが知られている。フェノールオキシダーゼは、モノフェノール及び／又はｏ−ジフェノールをアミンクロムに酸化し、メラニンの合成を開始する。 The ProPO system contains a complex sequence of proteins including pattern recognition proteins, ppA and proPO. In some arthropods, the proPO system has been described to be activated in the presence of small amounts of PAMP via a mechanism that has not yet been fully elucidated. In general, it is known that pro-ppA becomes active in the presence of PAMP and converts prophenol oxidase (inactive enzyme precursor) to active phenol oxidase by proteolytic attack. Phenol oxidase oxidizes monophenol and / or o-diphenol to amine chromium and initiates the synthesis of melanin.
米国特許第４９７０５２号（特許文献３０）には、フェノールオキシダーゼ活性又はｐｐＡペプチダーゼ活性を測定することによりＢＧ及びＰＧを検出するための、カイコの幼虫（ＳＬＰ）の血漿から得られる試薬の開発及び使用が記載されている。それはまた、ＢＧ又はＰＧの特異的測定を追求した試薬修飾を含む。この発明に基づき、米国特許第５５８５２４８号（特許文献３１）には、ｐｐＡ活性を検出するための合成の感受性発色基質の使用が記載されている。米国特許第６２７４５６５号Ｂ１（特許文献３２）では、ＢＧによって媒介される活性化経路を阻害することによって、ＰＧの特異的検出を可能とする試薬の修飾が保護されている。アッセイＳＬＰは、血小板における細菌汚染の検出に特に有用であると思われる（米国特許第７５９８０５４号Ｂ２（特許文献３３））。 US Pat. No. 4,970,052 describes the development and use of a reagent obtained from silkworm larvae (SLP) plasma to detect BG and PG by measuring phenol oxidase activity or ppA peptidase activity. Is described. It also includes reagent modifications that seek specific measurements of BG or PG. Based on this invention, US Pat. No. 5,585,248 describes the use of a synthetic sensitive chromogenic substrate to detect ppA activity. US Pat. No. 6,274,565 B1 (patent document 32) protects modifications of the reagent that allow specific detection of PG by inhibiting the activation pathway mediated by BG. The assay SLP appears to be particularly useful for detecting bacterial contamination in platelets (US Pat. No. 7,598,054 B2).
また、ｐｒｏＰＯ系を使用して、米国特許第６９８７００２号Ｂ２（特許文献３４）及び米国特許出願公開第２００２／０１９７６６２号Ａ１（特許文献３５）では、（１，３）−β−Ｄ−グルカンを検出及び定量するための、昆虫の幼虫（テネブリオ・モリトル（Ｔｅｎｅｂｒｉｏ ｍｏｌｉｔｏｒ）又はホロトリチア・ディオムファリア（Ｈｏｌｏｔｒｉｃｈｉａ ｄｉｏｍｐｈａｌｉａ））の全血リンパから得られた試薬を記載している。 In addition, using the proPO system, in US Pat. No. 6,987,002 B2 (Patent Document 34) and US Patent Application Publication No. 2002/0197662 A1 (Patent Document 35), (1,3) -β-D-glucan is obtained. Describes reagents derived from whole blood lymph of insect larvae (Tenebrio molitor or Horotrichia diomphalia) for detection and quantification.
イセエビのＰ．アーガス（Ｐ．ａｒｇｕｓ）におけるｐｒｏＰＯ系は血球において発見され［Ｐｅｒｄｏｍｏ−Ｍｏｒａｌｅｓ ｅｔ ａｌ．，Ｆｉｓｈ Ｓｈｅｌｌｆｉｓｈ Ｉｍｍｕｎｏｌ（２３）：１１８７−１１９５，２００７（非特許文献２１）］、かつ低濃度のＰＧ、ＢＧ及びＬＰＳの存在下において活性化可能である。ｐｒｏＰＯ酵素前駆体の活性化に関連する少なくとも２つのペプチダーゼがあり、一方はカルシウム依存性であり、他方はそうではない。ｐｒｏＰＯ系は、５ｋＤａのペプチダーゼ阻害剤及び正味の正電荷によって調節されている。系の活性成分から阻害剤を分離することにより、実質的にＰＧ、ＢＧ及びＬＰＳに対する応答の感度が増大する。ロブスターのｐｒｏＰＯ系はまた、血リンパの血漿画分に局在するＬＰＳ及びＢＧ認識タンパク質（ＬＧＢＰ）を含む。ＬＧＢＰは、ＬＰＳ及びＢＧに結合することにより活性化し、かつｐｐＡ活性の増加により溶解物中のフェノールオキシダーゼ活性を増加させることができる。 P. lobster The proPO system in P. argus was discovered in blood cells [Perdomo-Morales et al. , Fish Shellfish Immunol (23): 1187-1195, 2007 (non-patent document 21)], and can be activated in the presence of low concentrations of PG, BG and LPS. There are at least two peptidases associated with activation of the proPO enzyme precursor, one is calcium dependent and the other is not. The proPO system is regulated by a 5 kDa peptidase inhibitor and a net positive charge. Separating the inhibitor from the active component of the system substantially increases the sensitivity of the response to PG, BG and LPS. The lobster proPO system also contains LPS and BG recognition protein (LGBP), which are localized in the plasma fraction of the hemolymph. LGBP can be activated by binding to LPS and BG and can increase phenol oxidase activity in the lysate by increasing ppA activity.
本発明者らがここで初めて提示する組成物は、ロブスターの血リンパからの血球の水性抽出物であり、以下ロブスター血球溶解物（ＬＨＬ）と称する。ＬＨＬ中の関心対象の活性成分はｐｒｏＰＯ系であり、組成物はＬＰＳ、ＰＧ及びＢＧの検出及び定量を目的とするものである。本明細書においてロブスター（ｌｏｂｓｔｅｒ ｏｒ ｌｏｂｓｔｅｒｓ）という用語は、ザリガニ下目、イセエビ下目（イセエビ類）及びアナジャコ下目、歩行亜目（長尾類）、エビ目、甲殻綱、節足動物門の範囲内に記載の種を指している。 The composition that we present here for the first time is an aqueous extract of blood cells from lobster hemolymph, hereinafter referred to as lobster blood cell lysate (LHL). The active ingredient of interest in LHL is the proPO system and the composition is intended for the detection and quantification of LPS, PG and BG. In this specification, the term lobster or lobsters refers to the range of the crayfish lower lobster, lobster lobster (lobster) and octopus, gait suborder (long-tailed), lobster, crustacea, arthropoda Refers to the species described in.
ロブスターはまた、一部の地域において乱獲の対象であるが、多くの国（例えば、キューバ、ブラジル、オーストラリア、米国等）では主要な漁業資源を代表する非常に豊富な種である。血リンパは通常廃棄される副産物であるので、本組成物の生産は、食料として人間が消費するためのこれらの動物の入手可能性に影響を与えない。ロブスターは、最大の甲殻類のうちの１つであり、容易に入手可能な大量の血リンパを提供する。 Lobsters are also subject to overfishing in some areas, but in many countries (eg Cuba, Brazil, Australia, the United States, etc.) are very abundant species that represent major fishery resources. Since hemolymph is a by-product that is normally discarded, the production of the composition does not affect the availability of these animals for human consumption as food. Lobster is one of the largest crustaceans and provides a large amount of readily available hemolymph.
ＬＨＬを調製するために、血漿凝固を防止する、しかし好ましくは、血漿凝固並びに細胞の活性化及び凝集の両方を回避する適切な抗凝固剤を用いて、血リンパを引き抜く。抗凝固剤としては、メチルキサンチン誘導体、例えばテオフィリン、テオブロミン若しくはカフェイン、若しくはこれらの塩を含む等張液、又はシステインなどのスルフヒドリル基（−ＳＨ）を修飾可能な試薬、ヨードアセトアミド及びＮ−エチルマレイミドを使用することができる。また、適切な緩衝液によって提供されるｐＨ４．５−８の間を有するキレート剤を含有する溶液、例えば、変性アルセバー抗凝固剤（２７ｍＭのクエン酸ナトリウム、３３６ｍＭのＮａＣｌ、１１５ｍＭのグルコース、９ｍＭのＥＤＴＡ、ｐＨ７）又はクエン酸塩−ＥＤＴＡ抗凝固剤（０．４ＭのＮａＣｌ、０．１Ｍのグルコース、３０ｍＭのクエン酸三ナトリウム、２６ｍＭのクエン酸及び１０ｍＭのＥＤＴＡ、ｐＨ４．６）も適している。変性アルセバー抗凝固剤は、血リンパ：抗凝固剤が１：１（ｖ／ｖ）の割合で使用されることが好ましい。 To prepare LHL, the hemolymph is withdrawn with a suitable anticoagulant that prevents plasma clotting but preferably avoids both plasma clotting and cell activation and aggregation. Anticoagulants include methylxanthine derivatives such as isophylline, theobromine or caffeine, isotonic solutions containing salts thereof, or reagents capable of modifying sulfhydryl groups (-SH) such as cysteine, iodoacetamide and N-ethyl Maleimide can be used. Alternatively, a solution containing a chelating agent having a pH between 4.5 and 8 provided by an appropriate buffer, such as a modified Alsevere anticoagulant (27 mM sodium citrate, 336 mM NaCl, 115 mM glucose, 9 mM Also suitable are EDTA, pH 7) or citrate-EDTA anticoagulants (0.4 M NaCl, 0.1 M glucose, 30 mM trisodium citrate, 26 mM citric acid and 10 mM EDTA, pH 4.6). . The modified Alsever anticoagulant is preferably used in the ratio of hemolymph: anticoagulant 1: 1 (v / v).
血球を１０分間４℃にて７００ｇで遠心分離することにより血漿から分離する。血漿（上清）を廃棄し、沈降した血球を洗浄して残留血漿成分を除去する。この目的のために、血リンパ：抗凝固剤の最初の混合物に相当する体積以下の体積の抗凝固剤に血球を再懸濁し、続いて別の同一の遠心分離サイクルを行う。血球は少なくとも２回洗浄する必要がある。洗浄した血球ペレットを、好ましくは１〜１０ｍｌの間の体積の溶解緩衝液に懸濁させる。溶解緩衝液は、０．００１〜６００ｍＭの間の濃度のＮａＣｌ、酵素を安定化可能である薬剤（安定剤）、及び二価陽イオンを封鎖しない適した緩衝液によってもたらされる５〜８．５の間のｐＨを含み得る。５０ｍＭのＴｒｉｓ−ＨＣｌ、ｐＨ７．５、４５０ｍＭのＮａＣｌの溶解緩衝液が好ましい。 Blood cells are separated from plasma by centrifuging at 700 g for 10 minutes at 4 ° C. The plasma (supernatant) is discarded and the sedimented blood cells are washed to remove residual plasma components. For this purpose, the blood cells are resuspended in a volume of anticoagulant equal to or less than the volume corresponding to the initial mixture of hemolymph: anticoagulant, followed by another identical centrifugation cycle. Blood cells need to be washed at least twice. The washed blood cell pellet is preferably suspended in a volume of lysis buffer between 1 and 10 ml. The lysis buffer is 5 to 8.5 provided by NaCl at a concentration between 0.001 and 600 mM, an agent capable of stabilizing the enzyme (stabilizer), and a suitable buffer that does not sequester divalent cations. PH between. A 50 mM Tris-HCl, pH 7.5, 450 mM NaCl lysis buffer is preferred.
血球は、一般に細胞抽出物を調製するために生化学において使用される機械的、化学的又は酵素的であり得る破壊方法の中から適切な方法を用いて溶解させる。これらの細胞の特性を考慮して、最適な破裂方法には浸透圧ショック、凍結／融解、撹拌（ボルテックス）又は手動（Ｄｏｕｎｃｅ型及びＰｏｔｔｅｒ−Ｅｌｖｅｈｊｅｍｅｍ型）による均質化、及び超音波処理がある。血球ホモジネートを４℃で３０分間、１３０００ｒｐｍで遠心分離することで、清澄化した上清、すなわちＬＨＬを得る。 Blood cells are lysed using a suitable method from among the disruption methods that can be mechanical, chemical or enzymatic, commonly used in biochemistry to prepare cell extracts. Considering the characteristics of these cells, optimal rupture methods include osmotic shock, freeze / thaw, agitation (vortex) or manual (Dounce and Potter-Elvehjemem) homogenization, and sonication. The blood cell homogenate is centrifuged at 13000 rpm for 30 minutes at 4 ° C. to obtain a clarified supernatant, ie LHL.
本発明は、ＬＰＳ、ＰＧ及びＢＧに対するＬＨＬ応答の感度を高めるための修飾を含む。この修飾はＬＨＬに存在するペプチダーゼ阻害剤の除去又は不活性化に基づくものである。阻害剤は、分子のサイズ及び形状、電荷又は親和性に基づく分離技術によって除去することができる。好ましくは、サイズ排除、例えばゲル濾過クロマトグラフィ、限外濾過及びダイアフィルトレーションなどに基づく処置を用いる。ゲル濾過クロマトグラフィでは、１０〜６０ｋＤａの間、好ましくは３０ｋＤａの排除限界を有する樹脂を使用しなければならない。クロマトグラフィ分画は、３〜６０ｃｍ／時の間、好ましくは９ｃｍ／時の流速で実施し、適用する試料体積は、全カラム体積の１〜５％の間、好ましくは３％とすべきである。これらの条件下で、ｐｒｏＰＯ系の残留活性タンパク質は、カラムの空隙容量に溶出する。限外濾過及びダイアフィルトレーションを使用する場合、５〜６０ｋＤａの間、好ましくは１０〜４０ｋＤａの間のカットオフを有する膜を使用する。阻害剤を含まないＬＨＬは、以下、修飾ＬＨＬ（ＬＨＬ−Ｍ）と称する。 The present invention includes modifications to increase the sensitivity of the LHL response to LPS, PG and BG. This modification is based on the removal or inactivation of peptidase inhibitors present in LHL. Inhibitors can be removed by separation techniques based on molecular size and shape, charge or affinity. Preferably, treatments based on size exclusion such as gel filtration chromatography, ultrafiltration and diafiltration are used. In gel filtration chromatography, a resin having an exclusion limit of between 10-60 kDa, preferably 30 kDa must be used. Chromatographic fractionation should be performed at a flow rate between 3-60 cm / hr, preferably 9 cm / hr, and the sample volume applied should be between 1-5%, preferably 3% of the total column volume. Under these conditions, proPO-based residual active protein elutes in the void volume of the column. When using ultrafiltration and diafiltration, a membrane with a cutoff between 5 and 60 kDa, preferably between 10 and 40 kDa is used. LHL that does not contain an inhibitor is hereinafter referred to as modified LHL (LHL-M).
ＬＨＬ及びＬＨＬ−Ｍの両方の試薬は、ＰＧ、ＢＧ及びＬＰＳを検出するために使用される。この検出は、ロブスターにおけるｐｒｏＰＯ系の活性化に基づくものである（図１）。したがって、ＰＧ、ＢＧ及びＬＰＳの検出は、活性フェノールオキシダーゼ（ＦＯ）の酵素活性又はｐｐＡのペプチダーゼ活性を測定することにより行われる（図１）。 Both LHL and LHL-M reagents are used to detect PG, BG and LPS. This detection is based on the activation of the proPO system in lobster (FIG. 1). Therefore, detection of PG, BG and LPS is performed by measuring the enzymatic activity of active phenol oxidase (FO) or the peptidase activity of ppA (FIG. 1).
フェノールオキシダーゼ活性は、モノフェノール基質（例えばチラミン、Ｌ−チロシン、４−ヒドロキシフェニル酢酸、４−ヒドロキシフェニルプロピオン酸）又はジフェノール基質（例えばジヒドロキシ−Ｌ−フェニルアラニン（Ｌ−ＤＯＰＡ）、ドーパミン、３，４−ジヒドロキシフェニル酢酸、３，４−ジヒドロキシフェニルプロピオン酸、カテコール及びメチルカテコール）を用いて測定する。１．２ｍＭのドーパミンを用いて測定することが好ましい。着色生成物の形成により、フェノールオキシダーゼ活性を視覚的又は分光光度的に測定することが可能となる。 Phenol oxidase activity is measured by monophenol substrates (eg tyramine, L-tyrosine, 4-hydroxyphenylacetic acid, 4-hydroxyphenylpropionic acid) or diphenol substrates (eg dihydroxy-L-phenylalanine (L-DOPA), dopamine, 3, 4-dihydroxyphenylacetic acid, 3,4-dihydroxyphenylpropionic acid, catechol and methylcatechol). It is preferable to measure using 1.2 mM dopamine. The formation of a colored product allows the phenol oxidase activity to be measured visually or spectrophotometrically.
フェノールオキシダーゼ活性を測定するための方法の感度は、モノフェノール基質又はｏ−ジフェノール基質と３−メチル−２−ベンゾチアゾリンヒドラジン（ＭＢＴＨ）又はＢｅｓｔｈｏｒｎのヒドラゾンとを組み合わせることにより増大させることが可能である。ＭＢＴＨは、対応するアミンクロムよりもはるかに大きな消光係数又はモル吸光係数を有して、フェノールオキシダーゼにより生成されるｏ−キノンと安定した着色付加物（ＭＢＴＨ−キノン）を形成する強力な求核剤である［Ｅｓｐｉｎ ＪＣ ｅｔ ａｌ．，Ｅｕｒ Ｊ Ｂｉｏｃｈｅｍ，２６７：１２７０−１２７９，２０００（非特許文献２２）；Ｇａｒｃｉａ−Ｍｏｌｉｎａ Ｆ ｅｔ ａｌ．，Ｊ．Ａｇｒｉｃ．Ｆｏｏｄ Ｃｈｅｍ ５５：９７３９−９７４９，２００７（非特許文献２３）］。ＭＢＴＨは、０．３〜１５ｍＭ、好ましくは２〜７ｍＭの間の最終濃度で使用される。 The sensitivity of the method for measuring phenol oxidase activity can be increased by combining a monophenol substrate or o-diphenol substrate with 3-methyl-2-benzothiazoline hydrazine (MBTH) or Besthorn hydrazone. is there. MBTH has a much higher extinction coefficient or molar extinction coefficient than the corresponding amine chromium and is a strong nucleophile that forms a stable colored adduct (MBTH-quinone) with o-quinone produced by phenol oxidase. [Espin JC et al. , Eur J Biochem, 267: 1270-1279, 2000 (Non-Patent Document 22); Garcia-Molina F et al. , J .; Agric. Food Chem 55: 9739-9749, 2007 (Non-Patent Document 23)]. MBTH is used at a final concentration between 0.3-15 mM, preferably 2-7 mM.
ＢＧ、ＬＰＳ及びＰＧによって誘導されるｐｐＡのペプチダーゼ活性は、トリプシン様セリンプロテアーゼに対する発色基質又は蛍光発生基質を用いて測定する。これらの基質は、一般式Ｒ−Ｙ又はＲ−Ａｒｇ−Ｌｙｓ−Ｙであり、式中、Ｙはｐ−ニトロアニリン（ｐ−ＮＡ）のような発色団、又は発蛍光団、例えば７−アミド−４−メチルクマリン、ローダミン、若しくは７−アミド−４−トリフルオロメチルクマリンである。Ｒは保護基によりそのＮ末端が保護されたＬ若しくはＤアミノ酸、又は保護基によりそのＮ末端が保護されたＬ若しくはＤアミノ酸、若しくはこれらの組み合わせを含むペプチドを表す。 The peptidase activity of ppA induced by BG, LPS and PG is measured using a chromogenic or fluorogenic substrate for trypsin-like serine protease. These substrates are of the general formula RY or R-Arg-Lys-Y, where Y is a chromophore such as p-nitroaniline (p-NA), or a fluorophore such as 7-amide. -4-methylcoumarin, rhodamine, or 7-amido-4-trifluoromethylcoumarin. R represents a peptide containing an L or D amino acid whose N-terminus is protected by a protecting group, or an L or D amino acid whose N-terminus is protected by a protecting group, or a combination thereof.
ＬＨＬ又はＬＨＬ−Ｍ試薬を用いたＰＧ、ＬＰＳ及びＢＧを検出するためのアッセイは、速度論的、擬似速度論的又はエンドポイントの方法により行われ得る。反応は従来の分光光度計及び蛍光光度計を用いて検出可能である。しかしながら、より高い試料処理能力及び試薬の節約のために、可視光、蛍光の波長を読み取るように装備された、９６ウェルマイクロプレートを読み取ることが可能なマイクロプレートリーダ、又は両機能を実行可能なものを用いることが好ましい。 Assays for detecting PG, LPS and BG using LHL or LHL-M reagents can be performed by kinetic, pseudokinetic or endpoint methods. The reaction can be detected using conventional spectrophotometers and fluorometers. However, for higher sample throughput and reagent savings, a microplate reader capable of reading 96-well microplates equipped to read visible light, fluorescence wavelengths, or both functions can be performed It is preferable to use one.
速度論的アッセイは、基質を含む反応混合物の全成分を添加した直後の時間における生成物検出として定義される。擬似速度論的アッセイは、予めインキュベートした反応混合物への基質添加後に反応が測定されるものとして定義される。エンドポイントアッセイは、基質を用いた反応混合物の一定時間のインキュベーション後の反応の単一読み取りとして定義される。 A kinetic assay is defined as product detection at the time immediately after the addition of all components of the reaction mixture including the substrate. A pseudokinetic assay is defined as one in which the reaction is measured after addition of substrate to the pre-incubated reaction mixture. An endpoint assay is defined as a single reading of the reaction after a period of incubation of the reaction mixture with the substrate.
反応混合物は６〜９の間、好ましくは７．５のｐＨ値を有さなければならず、かつ二価の陽イオンを含んでも含まなくてもよい。カルシウム、マグネシウム又はマンガンを５ｍＭの最低濃度、好ましくは５０ｍＭ含む必要がある。試験温度は室温〜４５℃の間、好ましくは３７℃とすべきである。 The reaction mixture must have a pH value between 6 and 9, preferably 7.5, and may or may not contain a divalent cation. It is necessary to contain a minimum concentration of 5 mM of calcium, magnesium or manganese, preferably 50 mM. The test temperature should be between room temperature and 45 ° C, preferably 37 ° C.
本発明はまた、ＢＧ及びＬＰＳに対するＬＨＬ及びＬＨＬ−Ｍの両方の感度を増大させるためにＬＧＢＰの使用を含む。ＬＧＢＰは試薬の製剤中に含まれてもよく、又は反応混合物に後で添加してもよい。ＬＧＢＰは、ＬＰＳ及びＢＧを検出するためのアッセイにおいて、３〜２００μｇ／ｍｌ、好ましくは５０〜１２５μｇ／ｍｌの間の濃度範囲であり得る。 The present invention also includes the use of LGBP to increase the sensitivity of both LHL and LHL-M to BG and LPS. LGBP may be included in the reagent formulation or may be added later to the reaction mixture. LGBP may be in a concentration range between 3-200 μg / ml, preferably 50-125 μg / ml, in assays to detect LPS and BG.
ＬＧＢＰは血漿から得られ、それはまたＬＨＬ調製の豊富な副産物である。等電沈殿工程の後［Ｖａｒｇａｓ−Ａｌｂｏｒｅｓ Ｆ ｅｔ ａｌ．，Ｃｏｍｐ Ｂｉｏｃｈｅｍ Ｐｈｙｓｉｏｌ Ｂ Ｂｉｏｃｈｅｍ Ｍｏｌ Ｂｉｏｌ，１１６（４）：４５３−４５８，１９９７（非特許文献２４）］、ＬＧＢＰは（１，３）−β−Ｄ−グルカンに結合した親水性マトリックス［Ｖａｒｇａｓ−Ａｌｂｏｒｅｓ Ｆ ｅｔ ａｌ．，Ｃｏｍｐ Ｂｉｏｃｈｅｍ Ｐｈｙｓｉｏｌ Ｂ Ｂｉｏｃｈｅｍ Ｍｏｌ Ｂｉｏｌ，１１６（４）：４５３−４５８，１９９７（非特許文献２４）］、免疫親和性［Ｄｕｖｉｃ Ｂ ａｎｄ Ｓｏｄｅｒｈａｌｌ Ｋ，Ｊ Ｂｉｏｌ．Ｃｈｅｍ．，２６５（１６）：９３２７−９３３２，１９９０（非特許文献２５）］又はヘパリン［Ｊｉｍｅｎｅｚ−Ｖｅｇａｓ Ｆ ｅｔ ａｌ．，Ｆｉｓｈ Ｓｈｅｌｌｆｉｓｈ Ｉｍｍｕｎｏｌ １３（３）：１７１−１８１，２００２（非特許文献２６）］を用いた親和性クロマトグラフィにより精製可能である。好ましくは、ヘパリンをリガンドとして使用することで、単一のクロマトグラフィ工程で大量の純粋なＬＧＢＰを得ることが可能である。 LGBP is obtained from plasma, which is also a rich by-product of LHL preparation. After the isoelectric precipitation step [Vargas-Albores F et al. , Comp Biochem Physiol B Biochem Mol Biol, 116 (4): 453-458, 1997 (non-patent document 24)], LGBP is a hydrophilic matrix [Vargas-Albores bound to (1,3) -β-D-glucan. F et al. , Comp Biochem Physiol B Biochem Mol Biol, 116 (4): 453-458, 1997 (non-patent document 24)], immunoaffinity [Dubic B and Soderhall K, J Biol. Chem. , 265 (16): 9327-9332, 1990 (non-patent document 25)] or heparin [Jimenez-Vegas F et al. , Fish Shellfish Immunol 13 (3): 171-181, 2002 (Non-Patent Document 26)]. Preferably, by using heparin as a ligand, it is possible to obtain large amounts of pure LGBP in a single chromatography step.
（実施例） (Example)
ロブスター血球溶解物（ＬＨＬ）の調製
１０ｍｌの血リンパを、１０ｍｌの冷却抗凝固剤溶液を含む２０ｍｌの滅菌かつ発熱物質を含まない使い捨て注射器を使用して、第４歩行脚の基節から得た。使用した抗凝固剤は、２７ｍＭのクエン酸ナトリウム、３３６ｍＭの塩化ナトリウム、１１５ｍＭのグルコース、９ｍＭのＥＤＴＡ、ｐＨ７（１０００ｍオスモル）から成るアルセバー変性溶液であった。血リンパ：抗凝固剤の混合物を５０ｍｌの滅菌かつ発熱物質を含まないポリプロピレン遠心管に注ぎ、１０分間４℃で７００ｇにて遠心分離した。血漿を含有する上清を廃棄した。その後、ペレット化した血球を洗浄して残留血漿成分を除去した。これを達成するために、血球ペレットを２０ｍｌの冷却抗凝固剤に懸濁させ、次いで体積を元の抗凝固剤：血リンパの体積（５０ｍｌ）まで抗凝固剤を用いて上昇させ、上記のように再度遠心分離した。洗浄工程をもう一度繰り返した。洗浄した血球ペレットを、５０ｍＭのＴｒｉｓ−ＨＣｌ、４５０ｍＭのＮａＣｌ、ｐＨ７．５から成る３ｍｌの溶解緩衝液に再懸濁させ、次いで、１３ｍｍ×１０ｃｍのホウケイ酸チューブに移した。血球懸濁液を、氷浴中で１０秒間、２０ワットの３サイクルを用いて超音波処理により溶解させた。血球ホモジネートを３０分間４℃にて１３０００ｒｐｍで遠心分離することにより、清澄ＬＨＬを得た。
Preparation of Lobster Hemolysate (LHL) 10 ml hemolymph was obtained from the base of the 4th limb using a 20 ml sterile and pyrogen-free disposable syringe containing 10 ml of chilled anticoagulant solution. . The anticoagulant used was an Alsever denaturing solution consisting of 27 mM sodium citrate, 336 mM sodium chloride, 115 mM glucose, 9 mM EDTA, pH 7 (1000 mmol). The hemolymph: anticoagulant mixture was poured into 50 ml sterile and pyrogen free polypropylene centrifuge tubes and centrifuged at 700 g for 10 minutes at 4 ° C. The supernatant containing the plasma was discarded. Thereafter, the pelleted blood cells were washed to remove residual plasma components. To achieve this, the blood cell pellet is suspended in 20 ml of chilled anticoagulant and then the volume is raised with anticoagulant to the original anticoagulant: hemolymph volume (50 ml), as described above. Was centrifuged again. The washing process was repeated once more. The washed blood cell pellet was resuspended in 3 ml lysis buffer consisting of 50 mM Tris-HCl, 450 mM NaCl, pH 7.5 and then transferred to a 13 mm × 10 cm borosilicate tube. The blood cell suspension was lysed by sonication using 3 cycles of 20 watts in an ice bath for 10 seconds. The blood cell homogenate was centrifuged at 13000 rpm for 30 minutes at 4 ° C. to obtain clarified LHL.
ＬＨＬにおけるフェノールオキシダーゼ反応の感度に対するドーパミン濃度の影響とＬ−ＤＯＰＡ濃度の影響との比較
溶解物（０．１ｍｇ／ｍｌで４２０μｌ）を１ｍｇ／ｍｌのウシトリプシン４．２ｍｌを用いて３７℃で３０分間インキュベートした。この混合物の１００μｌを、５０ｍＭのＴｒｉｓ−ＨＣｌ、ｐＨ７．５中の種々の濃度のＬ−ＤＯＰＡ（０．３〜８ｍＭ）又はドーパミン（０．１５〜９ｍＭ）１５０μｌを含有するマイクロプレートのウェルに添加した。マイクロプレートリーダにおいて３７℃で２０分間、４９０ｎｍでフェノールオキシダーゼ活性を速度論的に測定した。
Comparison of the effect of dopamine concentration and L-DOPA concentration on the sensitivity of the phenol oxidase reaction in LHL The lysate (420 μl at 0.1 mg / ml) was used at 37 ° C. with 4.2 ml of 1 mg / ml bovine trypsin. Incubated for minutes. Add 100 μl of this mixture to the wells of a microplate containing 150 μl of various concentrations of L-DOPA (0.3-8 mM) or dopamine (0.15-9 mM) in 50 mM Tris-HCl, pH 7.5. did. Phenol oxidase activity was measured kinetically at 490 nm for 20 minutes at 37 ° C. in a microplate reader.
速度論的方法によるフェノールオキシダーゼ活性の検出を介した、ＬＨＬを用いたＰＧ、ＢＧ及びＬＰＳの測定
ＬＨＬ（１２ｍｇ／ｍｌ）を、５０ｍＭのＴｒｉｓ−ＨＣｌ、ｐＨ７．５を用いて０．５ｍｇ／ｍｌに希釈した。滅菌かつ発熱物質を含まないことが保証された９６ウェル平底マイクロプレートにおいてアッセイを実施した。反応混合物は１５０μｌの５０ｍＭ Ｔｒｉｓ−ＨＣｌ、ｐＨ７．５、５０ｍＭ ＣａＣｌ２、２０μｌのＬＨＬ、及び５０μｌの活性化物質（ＬＰＳ、ＰＧ又はＢＧ）から構成された。対照は、内毒素を含まない水であった。最後に、５０μｌの３．７５ｍＭドーパミンを基質として添加した。ドーパミンクロム形成は、マイクロプレートリーダで３７℃にて１５秒ごとに読み出すことによって、１時間の間、４９０ｎｍで速度論的に評価した。反応速度（ΔＯＤ４９０／分）は、ＯＤ４９０ｎｍ対時間のプロットの直線部分から決定した。あるいは、開始時間は反応混合物が０．１の吸光度値に到達するのに必要な時間として決定した。ＰＡＭＰ濃度の対数に対する開始時間の対数のプロットは線形であり、かつ定量目的のために有用であった。
Measurement of PG, BG and LPS with LHL via detection of phenol oxidase activity by kinetic method LHL (12 mg / ml) was 0.5 mg / ml with 50 mM Tris-HCl, pH 7.5. Dilute to The assay was performed in a 96 well flat bottom microplate that was guaranteed to be sterile and pyrogen free. The reaction mixture consisted of 150
ＬＨＬにおけるフェノールオキシダーゼ反応の増加に対するＭＢＴＨの効果。滴定
０．８ｍｇ／ｍＬのＬＨＬを、５０ｍＭのＴｒｉｓ−ＨＣｌ、ｐＨ７．５を用いて０．１ｍｇ／ｍＬに希釈した。アッセイは平底ウェルマイクロプレートにおいて実施した。反応混合物は４０μｌの５０ｍＭ Ｔｒｉｓ−ＨＣｌ、ｐＨ７．５、５０ｍＭ Ｔｒｉｓ−ＨＣｌ、ｐＨ７．５に溶解させた１６０μｌの１ｍｇ／ｍｌトリプシン、１０μｌのＬＨＬ及び蒸留水に溶解させた４０μｌのＭＢＴＨから構成された。この混合物を３７℃で２０分間インキュベートし、最後に５０μｌの３．７５ｍＭドーパミンを各ウェルに添加した。反応はマイクロプレートリーダにおいて３７℃で１時間の間、１５秒ごとに４９０ｎｍで速度論的に読み取った。
Effect of MBTH on increasing phenol oxidase reaction in LHL. Titration 0.8 mg / mL LHL was diluted to 0.1 mg / mL with 50 mM Tris-HCl, pH 7.5. The assay was performed in flat bottom well microplates. The reaction mixture consisted of 40
セリンペプチダーゼに対する発色基質を用いたＬＰＳに対するＬＨＬ応答の測定
溶解緩衝液中に塩化ナトリウムを含まない実施例１に記載した通りに得られる５０μｌのＬＨＬ（１ｍｇ／ｍｌ）を、１５０μｌの５０ｍＭ Ｔｒｉｓ−ＨＣｌ、ｐＨ７．５緩衝液、及び５０μｌのＬＰＳと合わせた。その後、５０μｌの０．６ｍＭ 発色基質Ｓ−２２２２（Ｂｚ−Ｉｌｅ−Ｇｌｕ（γ−ＯＲ）−Ｇｌｙ−Ａｒｇ−ｐＮＡ−ＨＣｌ）を各ウェルに添加した。放出されたｐ−ニトロアニリンを３７℃で１時間、４０５ｎｍで速度論的に検出した。
Measurement of LHL response to LPS using chromogenic substrate for
ゲル濾過クロマトグラフィによるプロテアーゼ阻害剤の分離
予め５０ｍＭのＴｒｉｓ−ＨＣｌ、０．２ＭのＮａＣｌ、ｐＨ７．５で平衡化したカラムＸＫ１６／７０（Ｖｔ＝１３１．４ｍｌ）中に充填したセファデックスＧ−５０スーパーファインでのゲル濾過クロマトグラフィを用いて、ＬＨＬ中のｐｒｏＰＯ活性化系の活性成分からプロテアーゼ阻害剤を分離する。４ｍｌの８ｍｇ／ｍｌ ＬＨＬ（３％Ｖｔ）を、０．３ｍｌ／分の流速で分画した。クロマトグラフィ分画を２８０ｎｍでモニタリングした。カラムの空隙容量に相当する画分を集め、Ｆ１又は修正ＬＨＬ（ＬＨＬ−Ｍ）と命名した。
Separation of protease inhibitors by gel filtration chromatography Sephadex G-50 Super packed in column XK16 / 70 (Vt = 131.4 ml) pre-equilibrated with 50 mM Tris-HCl, 0.2 M NaCl, pH 7.5 The protease inhibitor is separated from the active components of the proPO activation system in LHL using fine gel filtration chromatography. 4 ml of 8 mg / ml LHL (3% Vt) was fractionated at a flow rate of 0.3 ml / min. Chromatographic fractions were monitored at 280 nm. The fraction corresponding to the void volume of the column was collected and named F1 or modified LHL (LHL-M).
スピンカラムによるプロテアーゼ阻害剤の分離
ＬＨＬをセファデックスＧ−５０（１．５ｍｌ）で充填したスピンカラムで分画した。カラムを１分間１０００ｒｐｍにて４回洗浄することにより、カラムを５０ｍＭのＴｒｉｓ−ＨＣｌ、４５０ｍＭのＮａＣｌ、ｐＨ７．５緩衝液で平衡化した。実施例１の通りに調製した１５０μｌのＬＨＬをスピンカラムに適用し、１分間１０００ｒｐｍで遠心分離した。溶出液を集め、ＬＨＬ（Ｆ０画分とも称される）及び溶出液又は分画ＬＨＬ（Ｆ１又はＬＨＬ−Ｍとも称される）の両方を、タンパク質濃度、トリプシンに対する阻害活性、及びフェノールオキシダーゼ活性について分析した。
Separation of protease inhibitors by spin column LHL was fractionated on a spin column packed with Sephadex G-50 (1.5 ml). The column was equilibrated with 50 mM Tris-HCl, 450 mM NaCl, pH 7.5 buffer by washing the column 4 times at 1000 rpm for 1 minute. 150 μl of LHL prepared as in Example 1 was applied to a spin column and centrifuged at 1000 rpm for 1 minute. The eluate is collected and both LHL (also referred to as F0 fraction) and eluate or fraction LHL (also referred to as F1 or LHL-M) for protein concentration, trypsin inhibitory activity, and phenol oxidase activity. analyzed.
トリプシン阻害活性を欠損したＬＨＬ、すなわちＬＨＬ−ＭにおけるＬＰＳに対するフェノールオキシダーゼ反応の測定
阻害剤を含まない溶解物（修飾ＬＨＬ；ＬＨＬ−Ｍ）を実施例５に従って得た。５０μｌのＬＰＳ、１５０μｌのＴｒｉｓ−ＨＣｌ、５０ｍＭのＣａＣｌ２、ｐＨ７．５、及び２０μｌの０．８ｍｇ／ｍｌ溶解物（ＬＨＬ−Ｍ）の反応混合物を３０分間３７℃でインキュベートした。最後に５０μｌの３．７５ｍＭドーパミンを添加し、反応の進行を３７℃で１時間、４９０ｎｍで速度論的に読み取った。開始時間は、０．３の光学密度に到達するのに必要な時間として算出し、その後、ＬＰＳ濃度の対数に対する開始時間の対数をプロットした。
Measurement of phenol oxidase reaction to LPS in LHL lacking trypsin inhibitory activity, ie, LHL-M A lysate containing no inhibitor (modified LHL; LHL-M) was obtained according to Example 5. A reaction mixture of 50 μl LPS, 150 μl Tris-HCl, 50 mM CaCl 2 , pH 7.5, and 20 μl 0.8 mg / ml lysate (LHL-M) was incubated at 37 ° C. for 30 minutes. Finally 50 μl of 3.75 mM dopamine was added and the progress of the reaction was read kinetically at 490 nm for 1 hour at 37 ° C. The start time was calculated as the time required to reach an optical density of 0.3 and then the log of the start time against the log of the LPS concentration was plotted.
リポ多糖及びβ−１，３グルカン結合タンパク質（ＬＧＢＰ）の血漿からの精製
ＬＨＬ調製中に得られた血漿を、４℃で２０分間、５０００ｒｐｍで遠心分離して清澄化した。１００ｍｌの清澄化した血漿を、８０００Ｄａのカットオフの透析膜を用いて、４８時間４℃にて５Ｌの蒸留水に対して透析した。期間中、透析水を１２時間ごとに４回変更した。透析液を４℃で２０分間、５０００ｒｐｍで遠心分離し、上清を捨てた。タンパク質のペレットを５０ｍＭのＴｒｉｓ−ＨＣｌ、０．２ＭのＮａＣｌ、ｐＨ７．５（緩衝液Ａ）に懸濁させ、再度遠心分離して不溶性の変性タンパク質を除去した。５ｍｌ中の３５ｍｇの試料を、予め緩衝液Ａで平衡化したヘパリン−セファロースＣＬ−６Ｂを充填したカラムに適用した。非結合タンパク質を溶出後、カラムを緩衝液Ａ：緩衝液Ｂ（６０：４０ｖ／ｖ）の混合物の２．５カラム体積で洗浄した。緩衝液Ｂは５０ｍＭのＴｒｉｓ−ＨＣｌ、１ＭのＮａＣｌ、ｐＨ７．５から構成された。最後に、ＬＧＢＰを緩衝液Ａ：緩衝液Ｂ（３０：７０ｖ／ｖ）の５カラム体積で溶出させた。クロマトグラフィ工程を１ｍｌ／分の流速で実施し、タンパク質画分を２８０ｎｍで検出した。
Purification of Lipopolysaccharide and β-1,3 Glucan Binding Protein (LGBP) from Plasma Plasma obtained during LHL preparation was clarified by centrifugation at 5000 rpm for 20 minutes at 4 ° C. 100 ml of clarified plasma was dialyzed against 5 L of distilled water for 48 hours at 4 ° C. using a 8000 Da cutoff dialysis membrane. During the period, the dialysis water was changed 4 times every 12 hours. The dialysate was centrifuged at 5000 rpm for 20 minutes at 4 ° C., and the supernatant was discarded. The protein pellet was suspended in 50 mM Tris-HCl, 0.2 M NaCl, pH 7.5 (buffer A) and centrifuged again to remove insoluble denatured protein. A 35 mg sample in 5 ml was applied to a column packed with heparin-Sepharose CL-6B previously equilibrated with buffer A. After elution of unbound protein, the column was washed with 2.5 column volumes of a buffer A: buffer B (60:40 v / v) mixture. Buffer B consisted of 50 mM Tris-HCl, 1 M NaCl, pH 7.5. Finally, LGBP was eluted with 5 column volumes of buffer A: buffer B (30:70 v / v). The chromatography step was performed at a flow rate of 1 ml / min and the protein fraction was detected at 280 nm.
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| EP2811031B8 (en) | 2018-12-26 |
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| BR112014016111B1 (en) | 2020-11-24 |
| JP2015506679A (en) | 2015-03-05 |
| US20140356889A1 (en) | 2014-12-04 |
| IN2014DN05849A (en) | 2015-05-22 |
| BR112014016111A8 (en) | 2020-10-27 |
| CA2861982C (en) | 2020-11-10 |
| CU20110243A7 (en) | 2013-08-29 |
| US10844421B2 (en) | 2020-11-24 |
| CA2861982A1 (en) | 2013-08-08 |
| CU24073B1 (en) | 2015-01-29 |
| US20180340206A1 (en) | 2018-11-29 |
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